As a method of extracting synthesized compounds as a single component, generally, a method of using a preparative liquid chromatograph, among liquid chromatographs, and performing separation/fractionation, and then performing concentration is used.
With respect to concentration, although a vacuum evaporator is also used, concentration by a trap column is used often because, if an elimination column is used in combination, additives and the like may be eliminated (see Patent Document 1).
Generally, in the case of trapping a sample in a trap column, if the sample concentration is high, or the solvent strength of a sample solvent is high, trapping is difficult, and thus, a diluted sample is delivered to the trap column so as to facilitate trapping.
Dilution may be performed at the time of adjusting the sample, but in the case where the sample volume after dilution is excessively high, processing is difficult, and thus, dilution is performed online. In online dilution, a flow path similar to the flow path structure of a sample concentration device as shown in FIGS. 1 and 4 is used as an embodiment.
In FIG. 1, a high-pressure valve 2 is switched to an injection position according to which a sample loop 4 is incorporated in a main flow path including a sample push unit 5 and a trap column 8, and in FIG. 4, the high-pressure valve 2 is switched to a metering position according to which the sample loop 4 is incorporated to a metering flow path including a syringe pump 3 and a sampling needle 30.
According to such a sample concentration device, the high-pressure valve 2 is switched from an idling state (the high-pressure valve 2 is in the state in FIG. 1, which is the same as the injection position) to a sample metering state in FIG. 4, and a sample is drawn by the syringe pump 3 at an autosampler 1 and is introduced into the sample loop 4. Next, the high-pressure valve 2 is switched to the injection state in FIG. 1, and the sample in the sample loop 4 is pushed out from the sample loop 4 by a mobile phase delivered from the sample push unit 5. Then, the sample is diluted by being mixed with a diluent (normally, of the same composition as the mobile phase) delivered from the make-up unit 6 at the three-way joint 7 on the main flow path, and is lead to, and trapped in, the trap column 8.
The dilution rate at this time is normally several times the sample volume. The make-up unit 6 is expected to compensate for the flow rate of (dilution rate−1) times the flow rate of the sample push unit 5 at least until the sample passes through the three-way joint 7. After the sample passes through the three-way joint 7, the sample push unit 5 is expected to perform delivery until the sample is completely fixed to the trap column 8.
Since separation/analysis are not performed by the sample concentration device, normally, a mobile phase does not have to be delivered from the sample push unit 5 at the time of idling, and the make-up unit 6 does not have to perform delivery either. Especially, in the case of performing concentration of a large volume of sample by a large volume of mobile phase and a large volume of diluent, it is desirable to start delivery immediately before concentration is performed, and to immediately stop delivery when concentration is complete. However, in reality, it is not easy to control the delivery operation in this manner due to the following circumstances.
An example of a sample that is used by such a sample concentration device is a single component sample obtained by separating synthesized compounds by a preparative system. As an example, the fractionation for two component peaks according to the preparative system are shown in FIG. 3. In this example, the peak of a component B (retention time: 5.8 min) overlaps a tailing component A (retention time: 5.7 min). When a flow rate of 100 mL/min and a maximum fraction volume of 10 mL for one fraction vial are given as conditions, the upper limit of the volume of the vial is reached in 0.1 minutes. Then, in this example, it can be seen that when a fraction 9 reaches the maximum fraction volume 10 mL of a vial #1, it is switched to a fraction 10, and then, before the maximum fraction volume 10 mL of a vial #2 is reached, the rise of a peak of the component B is detected and switching to a fraction 11 is performed, and switching to a fraction 12 takes place when a fall of the peak of the component B is detected.
In the example in FIG. 3, the fractions 9, 10 and 12, which are a single component, are taken as a sample, and are injected to a concentration system based on a trap column, and concentration is performed.
With a general liquid chromatography analysis system, sample injection and the analysis process are programmed according to a table called a batch table (referred to also as a sequence table or a sample set). One row of the batch table corresponds to one analysis, and each row contains the position of a sample vial, the sample volume, and other analysis conditions.
Other analysis conditions include an analysis initial parameter set, a pre-injection processing program for customizing the operation of the autosampler 1 (normally referred to as a pre-treatment program. In the case of not performing customization, a standard pre-treatment operation is internally programmed), a time program (also referred to as an event or an event program), and the like, and normally, these are stored in a parameter set called an instrument method. (The pre-treatment program may be specified in the batch table independently of the instrument method.)
The process in one row of the batch table is performed in the order of downloading of the position of a sample vial, the sample volume and other analysis conditions to the device (download), execution of the pre-treatment program (pre-treatment execution), and execution of a time program and start of recording of a chromatogram (analysis execution).
As programming methods of the example in FIG. 3, there are the following (a) to (d).
(a) The fractions 9, 10 and 12 are transferred to one vial and are taken as one sample, and programming is performed with “one row/one injection”.
The “injection” here refers to connection of the sample loop 4 to the main flow path by switching of the high-pressure valve 2 to the injection position after one sample is introduced into the sample loop 4 by the pre-treatment program. In many cases, an event signal (an electrical signal, a communication command, a software instruction) for performing recording start of a chromatogram or a time program is output simultaneously with the switching of the high-pressure valve 2, but the event signal may be output before or after the switching of the high-pressure valve 2.
In this case, according to the instrument method, an “injection” process for one sample is performed by the pre-treatment program, and the sample push unit 5 and the make-up unit 6 are controlled by the time program, and fixation of the sample to the trap column 8 is performed. Here, after the sample is introduced into the sample loop 4, and before the high-pressure valve 2 is switched, an event signal is output to start delivery of a mobile phase and a diluent by the sample push unit 5 and the make-up unit 6 by the time program, and switching of the high-pressure valve 2 is performed after the delivery becomes stable.
(b) Programming is performed assuming “three rows/two loadings, one injection” for the fractions 9, 10 and 12.
The “loading” here means that the high-pressure valve 2 is not switched after a sample is introduced into the sample loop 4 by the pre-treatment program. In the case of this example, one row corresponds to one loading, and thus, after the “loading”, an event signal is output by the pre-treatment program and the time program is started, but since the sample push unit 5 and the make-up unit 6 do not have to operate, the time program is ended swiftly. Additionally, since the time program is ended swiftly, the autosampler 1, which cannot output an event signal unless the high-pressure valve 2 is switched, may perform a process corresponding to the “loading” in the “injection” process, but detailed description here is omitted.
In reality, an instrument method for sample push for until the N−1th row and an instrument method for injection for the Nth row are prepared and combined. In the case of this example, “loading” of one sample is performed by the pre-treatment program by the instrument method for sample push for the fractions 9 and 10, and “injection” of one sample and fixation of the sample to the trap column 8 are performed by the pre-treatment program by the instrument method for injection for the fraction 12. The contents of the processing by the instrument method for injection are the same as the contents of the processing by the instrument method of (a). Here, the samples of the fractions 9 and 10 are sent to the trap column 8 together with the sample of the fraction 12.
(c) Programming is performed assuming “one row/two loadings” for the fraction 9 with fixed volume and the fraction 10 with variable volume, and assuming “one row/one injection” for the fraction 12 with variable volume.
In reality, an instrument method for sample push of “one row/N loading (N: 1 or more, N−1 fraction: fixed volume, one fraction: variable volume)” and an instrument method for injection of “one row/one injection” are prepared and combined. In the case of this example, “loading” of one sample with fixed volume and one sample with variable volume (the volume is specified in the batch table) is performed by the pre-treatment program by the instrument method for sample push for the fractions 9 and 10, and “injection” of one sample with variable volume (the volume is specified in the batch table) and fixation of the sample to the trap column 8 are performed by the pre-treatment program by the instrument method for injection for the fraction 12. The contents of the processing by the instrument method for injection are the same as the contents of the processing by the instrument method of (a).
(d) Programming is performed assuming “one row/two loadings, one injection” for the fractions 9, 10 and 12, and “loading” and “injection” are performed by the pre-treatment program of the autosampler included in the instrument method by separately customizing the volumes of the fraction 9, 10 and 12.
According to this method, the instrument method performs, by the pre-treatment program, “loading” of each of the samples of the fractions 9 and 10, and “injection” of the sample of the fraction 12, and performs fixation to the trap column 8. The contents of the processing after introduction into the sample loop 4 are the same as the contents of the processing by the instrument method of (a).
In any of (a) to (d), the make-up unit 6 has to deliver a diluent by the volume several times the actual sample injection volume.
Here, the “actual sample injection volume” that is introduced into the sample loop 4 and injected into the main flow path is the total sample volume in the case where no special pre-treatment is required, and is the volume obtained by adding the volume of an additive to the total sample volume in the case where pre-treatment such as mixing of an additive is to be performed, and specification thereof in advance is difficult. Accordingly, instead of the “actual sample injection volume”, normally, the “maximum sample injection volume” is defined based on the volume of the sample loop 4 and the number of times of injection into the main flow path, and the maximum fraction volume and the maximum number of fraction vials.
The main objects of a liquid chromatography analysis are separation and analysis, and the retention time of a sample is not dependent on the sample volume, and only the tailing factor determining the elution range of the sample is dependent on the sample volume. Accordingly, in the case of using a specific sample, the analysis time at the liquid chromatography analysis may be assumed to be constant without depending on the sample volume.
Additionally, the tailing factor in separation/analysis is an index defined as W0.05/2f, and the greater tailing factor means greater tailing. Here, W0.05 is the peak width at the height which is 5% of the peak height, and f is the distance, within W0.05, from the rise of the peak to the top of the peak.
With the device control of a liquid chromatography analysis device, an operation may be programmed in the instrument method based on the analysis time and the time program (referred to also as an event or an event program), but control of operating a solvent delivery pump based on the actual sample injection volume cannot be performed.
Accordingly, with the liquid chromatography analysis device, in the case of performing trap concentration, a method of controlling the pump by switching the instrument method prepared for each sample volume range, or a method of controlling the pump by a time calculated with respect to the maximum sample injection volume is used.